Use of botulinum toxin therapy for urinary incontinence and related disorders

ABSTRACT

The present invention relates to methods for treating neurological-urological conditions. This is accomplished by administration of at least one neurotoxin.

FIELD OF THE INVENTION

[0001] The present invention provides methods for treatingneuronally-mediated urologic and related disorders, for example, benignprosatic hyperplasia (BPH) and prostatitis. This is accomplished byadministering a composition that includes at least one neurotoxiccompound or by conventional therapies.

BACKGROUND OF THE INVENTION

[0002] Many medical conditions in urology are rooted in a spasticdysfunction of the sacral reflex arcs. Examples of such conditionsinclude pelvic pain (e.g., interstitial cystitis, endometriosis,prostatodynia, urethral instability syndromes), pelvic myofascialelements (e.g., levator sphincter, dysmenorrhea, anal fistula,hemorrhoid), urinary incontinence (e.g., unstable bladder, unstablesphincter), prostate disorders (e.g., BPH, prostatitis, prostatecancer), recurrent infection (secondary to sphincter spasticity), andurinary retention (secondary to spastic sphincter, hypertrophied bladderneck) and neurogenic bladder dysfunction (e.g., Parkinson's Disease,spinal cord injury, stroke, multiple sclerosis, spasm reflex).

[0003] The prostate is a partially glandular and partially fibromusculargland of the male reproductive system. During aging, the prostate tendsto enlarge (hypertrophy). This prostatic enlargement can lead tourethral obstruction and voiding dysfunction.

[0004] Prostatic enlargement is a common occurrence in older men. Lyttonet al. (Lytton, B., Emery, J. M. and Harvard, B. M. [1973] 99: 639-645)estimated that a 45 year old male had a 10% risk of prostate surgery byage 70. The U.S. Census Report estimates that there are 30 millionpeople today over age 65. This segment of the population is projected torise to 50 million over the next 30 years. Therefore, the number of menwith prostatic enlargement also will increase. According to draftreports of the National Kidney and Urologic Disease Advisory Board,425,000 prostatectomies were performed in the United States in 1989.Based on population growth estimates, the number of prostatectomiesperformed annually will rise to 800,000/year by the year 2020.

[0005] The urethra passes through the prostate (prostatic urethra) as itcourses to the external urethral orifice. The prostate has five distinctlobes that are apparent at 12 weeks in the human fetus (Lowsley, O. S.Am. J. Anat. [1912] 13: 299-349.). Although the lobular branching foundin the fetus is not visible in the prepubescent prostate, the lateralmiddle anterior and posterior lobes are used to describe the enlargedprostate.

[0006] A more recent viewpoint is that the prostate also is comprised ofseveral morphologically distinct zones (McNeal J. Urol. Clin. North Am.[1990] 17(3): 477-486). The majority of the glandular volume is composedof the peripheral zone (˜70-75%). The remainder of glandular volume isdivided into the central zone (˜20-25%), the transition zone (˜5-10%)and the periurethral glandular zone (˜1%).

[0007] McNeal (1990) reported that BPH develops in the transition zoneand the periurethral glandular zone. BPH nodules develop either withinor immediately adjacent to the pre-prostatic sphincteric zone. Thetransition zone is a small region close to the urethra intimatelyrelated to the proximal urethral sphincter. The stroma of the transitionzone is dense and compact, and is unusually susceptible toneurologically-induced disturbances of growth control. Its glandspenetrate the sphincter, while sphincter muscle fibers penetrate thetransition stroma. The periurethral glandular zone has a similarurogenic sinus origin as the transition zone.

[0008] BPH may be associated with increased amounts of stroma relativeto epithelium (Bartsch, G., Muller, H R., Oberholzer, M., Rohr, H., P.,J. Urol. [1979] 122: 487-491). A significant portion of the stroma issmooth muscle (McNeal, 1990) which is under sympathetic nervous control.The contractile properties of this smooth muscle could account for thedynamic component of obstruction in BPH (Bruschini, H. et al. [1978]Invest. Urol. 15(4): 288-90; Lepor, H. [1990] Urol. Clin. North Am.17(3): 651-658).

[0009] In addition to sympathetic control of prostatic stroma, theprostate is highly innervated. The prostate nerve fibers enter theprostate from the posterior lateral aspect, with a concentration ofganglia near the junction between the prostate and the seminal vesicles(Maggi, C. A., ed. [1993] Nervous control of the Urogenital System,Harwood Academic Publishers; Higgins, J. R. A. and Gosling, J. A. [1989]Prostate Suppl. 2: 5-16). Acetylcholine (ACH), neuropeptide Y (NPY),vasoactive intestinal peptide (VIP) and noradrenaline fibers have beendescribed in this gland. A rich plexus of ACH-positive nerve cell bodiesis associated with secretory acini in all parts of the gland. Some ofthe ACH fibers also contain NPY neurons. VIP-containing neurons havebeen found associated with ACH-containing nerve cell bodies. Occasionalneurons have been found between the ACH-staining nerve fibers,suggesting that both NPY and noradrenergic neurons supply smooth muscle(Higgins, J. R. A. and Gosling, J. A. [1989] Prostate Suppl. 2: 5-16).

[0010] Autonomic nerves are distributed evenly between the central andperipheral zones of the prostate (Higgins, J. R. A. and Gosling, J. A.[1989] Prostate Suppl. 2: 5-16). Peripheral neuronal control is similar.In addition, there is no difference in the type of nerve fibers foundassociated with either epithelial or stromal elements of the gland.

[0011] The anatomical studies of nerve fiber types in the prostate,coupled with other studies of innervation of prostatic stroma (BrushingH., Schmidt, R. A., Tanagho, E. A., [1978] Invest. Urol. 15(4): 288-290;Watanabe, H., Shima, M., Kojima, M., Ohe, H. L. [1989] Pharmacol. Res.21(Suppl 2): 85-94) suggest that cholinergic innervation influencesepithelial behavior, while adrenergic innervation influences stromaltonus (excitability). These observations have provided a rationale forthe use of for example, alpha blockers in the treatment of BPH. Theeffects of alpha blockers (Downie, J. W. and Bialik, G. J. [1988] J.Pharmacol. Exp. Ther. 246(1): 352-358) can also account for improvementsin symptoms of BPH as a result of dampening of dysfunctional striatedsphincter behavior by the alpha blockers.

[0012] Studies have also shown that there are several tachykinins (forexample, substance P [SP], calcitonin gene related peptide [CGRP],neurokinin A, bradykinin, and nerve growth factor [NGF]) that caninfluence the tonus of smooth muscle (Hakanson, et al., [1987]Neuroscience 21(3): 943-950). Neurotransmitter receptors have beenquantified throughout the prostate (e.g., NPY, VIP, SP, leu-enkephalin(L-enk), met-enkephalin, 5-HT, somatostatin, acetylcholinesterasepositive fibers (ACTH), and dopamine beta-hydroxylase (DBH) (Crowe, R.,Chapple, C. R., Burnstock, G. The Human Prostate Gland: A Histochemicaland Immunohistochemical Study of Neuropeptides, Serotonins, Dopaminebeta-Hydroxylase and Acetylcholinesterase in Autonomic Nerves andGanglia). There is some variation in receptor density at differentprostatic sites in benign prostatic hyperplasia.

[0013] Changes in electrophysiologically recorded cellular behavior andin concentration of neuropeptides within the spinal cord have been shownto be a secondary consequence of mechanical pinch to the tail muscles ofa rat, catheter stimulation of the posterior urethra, andelectrostimulation of a peripheral nerve. Dyssynergia between thedetrusor and the urethral sphincter is a significant finding inprostatodynia patients. Denervation of the prostate has been shown toproduce dramatic changes within the prostatic epithelium. Thus there isevidence that experimentally induced alterations in neurologicalinfluences can be produced in the sacral, spinal cord, bladder orurethra through mechano-, electro-, chemical or thermal (microwave,laser) methods to change irritative behavior. However, there have beenno known attempts to use neurotoxins for therapeutic applications.

[0014] There is poor correlation between the degree of prostaticenlargement and the severity of symptoms. While 80% of men age 70 showBPH on transrectal ultrasound scans, only 20% seek surgery (Coffey, D.S. and Walsh, P. C. [1990] Urol. Clin. North Am. 17(3): 461-475), thetreatment of choice for BPH (Fowler, F. J. Jr., Wennberg, J. E.,Timothy, R. P. [1988] J. Amer. Med. Assoc., 259(20): 3022-3028).Symptoms of irritation may far exceed symptoms expected based on thesize of the prostate. Symptoms may improve after surgical treatment ofBPH by procedures such as transurethral resection of the prostate (TURP)(Christensen, Aagaard, M. M. J., Madsen, P. O. [1990] Urol. Clin. NorthAm. 17(3): 621-629), balloon dilation (Dowd, J. B. and Smith, J. J. III[1990] Urol. Clin. North Am. 17(3): 671-677), or prostatic hyperthermia(Baert, L., Ameye, F., Willemen, P., et al., [1990] J. Urol. 144:1383-1386). However, symptoms persist in as many as 15% of all BPHpatients (Baert, L., Ameye, F., Willemen, P., et al., [1990] J. Urol.144: 1383-1386; Wennberg, J. E., Mullly, A. G., Hanley, D., Timothy, R.P., Fowler, F. J., Roos, R. P., Barry, M. J. et al., [1988] J. Amer.Med. Assoc. 259: 3027-3030). Up to 25% of BPH patients have secondaryprocedures in long term follow-up studies, suggesting that surgicalapproaches do not address the fundamental mechanisms that produce BPH,i.e., the faulty neurological influence (control mechanism) on theintegrity of the lower unary tract.

[0015] The need for repeated surgeries, the morbidity and mortalityassociated with TURP and the cost of surgery have led to the developmentof some non-surgical approaches such as androgen ablation (McConnell, J.D., [1990] Urol. Clin. North Am. 17(3): 661-670) and the use of alphablockers discussed above, but few medical or surgical treatments to datehave produced a restoration of void behavior to normal state (flow rateof about 25 cc/sec and void volume of about 400 cc).

[0016] The present invention uses chemical and non-chemical methods,particularly neurotoxins, to modulate neuronally-mediated urologic andrelated disorders. For example, such methods can be used to treat BPHand related conditions such as prostatitis. The instant invention alsomay remove triggers of changes in the CNS by non-chemical methodsincluding biofeedback, or by chemical methods that treat BPH and otherurological conditions by the administration of substances that blockvarious neurological activities, such as, for example, selectedneurotoxins.

OBJECTS AND SUMMARY OF THE PRESENT INVENTION

[0017] It is an object of the instant invention to provide safe,inexpensive, out patient methods for the prevention and treatment ofurological-neurological dysfunctional states or conditions, for example,prostatic enlargement.

[0018] It is a further object of the present invention to providecompositions for this therapeutic goal. It is a still further object ofthe present invention to provide dosages and methods of administrationfor compositions useful for the prevention and treatment ofneurological-urological conditions.

[0019] Other objects of the present invention will be readily apparentto those of ordinary skill in the art.

[0020] In accordance with one aspect of the present invention, there areprovided methods of treating urological-neurological conditions inmammals, said methods comprising the step of administering atherapeutically effective amount of at least one neurotoxin to such amammal. It is preferred that the neurotoxin inhibits synaptic function.Such inhibition produces selective denervation, and, for example,atrophy of the prostate and reversal of irritative symptoms associatedwith prostatic enlargement. In one embodiment of the instant invention,the neurotoxin induces dysfunction of the presynaptic neuronal terminalby specific binding and blockade of acetylcholine release at myoneuraljunctions. Such a neurotoxin can be, for example, botulinum toxin type A(Botox, Allergen).

[0021] Preferably, the neurotoxin is safe, highly selective and easy todeliver, including when combined with other therapies. Other usefulneurotoxins include capsaicin, resinoferatoxin and α-bungotoxin.Delivery of the neurotoxin can be by any suitable means. A convenientand localized method of delivery is by injection.

[0022] A therapeutically effective amount of the neurotoxin is thedosage sufficient to inhibit neuronal activity for at least one week,more preferably one month, most preferably for approximately 6 to 8months or longer. Dosing can be single dosage or cumulative (serialdosing), and can be readily determined by one skilled in the art.Neurotoxin can be delivered serially (i.e., one time per month, one timeper every six months) so that the therapeutic effect can be optimized.Such a dosage schedule is readily determined by one skilled in the artbased on, e.g., patient size and the condition to be treated, and willdepend on many factors, including the neurotoxin selected, the conditionto be treated, the degree of irritation, and other variables. Onesuggested course of treatment for BPH is 200 units every three days upto the LD50 for Botox or about 2500 units.

[0023] The aforementioned methods of treatment should be particularlyuseful for the long-term control of neurological-urological disorders,e.g., the symptoms of prostatic enlargement, without the need forsurgical intervention. Furthermore, the methods of the instant inventionprovide for control of neurological-urological disorders, e.g., BPH andrelated conditions, in a highly selective manner, without the potentialside effects and treatment failures associated with current treatmentmodalities.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

[0024] “Urological-neurological condition or disorder” includes manymedical conditions in urology rooted in a spastic dysfunction and/ordegeneration of the sacral reflex arcs. Examples of such conditionsinclude pelvic pain (e.g., interstitial cystitis, endometriosis,prostatodynia, urethral instability syndromes), pelvic myofascialelements (e.g., levator sphincter, dysmenorrhea, anal fistula,hemorrhoid), urinary incontinence (e.g., motor or sensory, unstablebladder, unstable sphincter), prostate disorders (e.g., BPH, prostatecancer), recurrent infection (secondary to sphincter spasticity), andurinary retention (secondary to spastic sphincter, hypertrophied bladderneck), and neurogenic bladder dysfunction (e.g., Parkinson's Disease,spinal cord injury, stroke, multiple sclerosis, spasm reflex) and othersuch urological conditions of a nervous etiology. The prostaticenlargement that can be treated according to the methods of the instantinvention can be of any etiology. The instant invention is particularlysuited for the treatment of prostatic hyperplasia, especially benignprostatic hyperplasia. The present invention can also be used for thetreatment of enlargement of the prostate with inflammation(prostatitis), particularly abacterial prostatitis. In addition, themethods of the instant invention can be used for the treatment ofprostatodynia.

[0025] Without being bound by theory, the basis for the treatment of theneurological-urological conditions according to the instant invention isthe removal or modulation of the neural basis for the dysfunctionalregulation of the affected tissue. For example, the modulation of theneural basis of prostate glandular dysfunction can be accomplished byany non-surgical means known in the art. Such means can include, forexample, biofeedback, α-blockers, pharmacological methods, and the useof one or more neurotoxins to inhibit synaptic function in the affectedgland. It is preferred that the neurotoxin cause long-lasting inhibitionof synaptic function, preferably greater than one week, more preferablygreater than one month, most preferably six to eight months or longer.Such neurotoxins can include, for example, capsaicin, resinoferatoxin,α-bungotoxin, terodotoxin and botulinum toxin. Botulinum toxin is apreferred neurotoxin according to the instant invention, particularlybotulinum toxin A, more particularly Botox (Allergen).

[0026] The toxin can be formulated in any pharmaceutically acceptableformulation in any pharmaceutically acceptable form. Such forms andformulations include liquids, powders, creams, emulsions, pills,troches, suppositories, suspensions, solutions, and the like. The toxincan also be used in any pharmaceutically acceptable form supplied by anymanufacturer.

[0027] In a preferred embodiment in accordance with the method of theinstant invention, the neurotoxin is botulinum toxin type A.Therapeutically effective amounts of botulinum toxin can be any amountsor doses that are less than a toxic dose, for example, less than about3000 IU/70 kg male, preferably between 100 IU/70 kg male to 1200 IU/70kg. The dosages can be given as a single dose, or as divided doses, forexample, divided over the course of four weeks.

[0028] The neurotoxins of the instant invention can be administered byany suitable means. In the preferred embodiment of the invention,botulinum toxin is administered by injection. Such injection can beadministered to any affected area. For example, the neurotoxin can beinjected urethroscopically into the prostate with 200 IU with single orserial dosing. Preferably, the neurotoxin is injected every three daysuntil a therapeutic effect is achieved or up to about 2500 units.

[0029] The following techniques are used in this invention:

[0030] Tissue Preparation for Light Microscopy

[0031] Tissues are fixed in 6% paraformaldehyde in 0.1 M phosphatebuffer, pH 7.2, for 24 hours, dehydrated in graded alcohol and xylene,and embedded in paraffin. Sections are cut and stained with appropriatestains, such as hematoxylin/eosin.

[0032] Tissue Preparation for Electron Microscopy

[0033] Tissues are collected and fixed in 2.5% glutaraldehyde in 0.1 Mphosphate buffer, pH 7.2, for 1 hour at 4° C., then incubated with 0.1%osmium tetroxide for 1 hour and embedded in EPON. Ultrathin sections (80nm) are prepared and stained with lead citrate/uranyl acetate andexamined with an electron microscope (Philips, model 201).

[0034] Tunel Stain for Apoptosis

[0035] The tissue is fixed and embedded as described above. The tissuesare deparaffinized and reacted with Proteinase K (Boehringer). They arefurther treated with peroxidase and TDT enzyme and placed in ahumidifier set at 37° C. for one hour. The sections are washed andanti-digoxigenin-peroxidase is added for 30 minutes, followed bystaining with nickel-DAB (diaminobenzene).

[0036] Immunohistochemistry Studies

[0037] The presence of the neuropeptides VIP, SP, NPY, L-Enk andcalcitonin gene-related peptide (CGRP) as well as the expression oftransforming growth actor beta (TGF-beta), transforming growth factoralpha (TGF-alpha), epidermal growth factor (EGF) and basic fibroblastgrowth factor (bFGF) are determined in prostatic tissues usingappropriate monoclonal antibodies. Use of neurotoxins results inprostatic atrophy, which should be reflected by lower levels of growthfactors in treated prostatic tissue.

[0038] Sections are incubated overnight at room temperature with primaryantibodies followed by immunostaining with avidin-biotin-peroxidase(Vectastain Elite ABC, Vector Labs, USA). Rabbit polyclonal antiserumagainst the neurotransmitters VIP, CGRP, SP, NPY and L-Enk (PeninsulaLabs, USA) is used in these preparations, at dilutions of 1:8000 to1:12,000. Immunocytochemical controls consist of preabsorbing theprimary antiserum with appropriate antigen, or their substitution withnormal serum (Blasi J., Chapman, E. R., Yamaskai S., Binz, T., Niemann,H and Jahn, R. [1993] The EMBO Journal 12: 4821-4828; Black, J. D. andDolly, J. O. [1986] J. Cell Biol. 103; 535-544; Linial, M. [1995] Is. J.Med. Sci. 31: 591-595). After mounting on slides, sections arecounterstained with eosin, dehydrated and coverslipped.

[0039] Western Blot Analysis of Growth Factor Expression

[0040] Treated and untreated prostate cell homogenates are examined forexpression of growth factors by Western blot analysis. Cell homogenateprotein is separated by electrophoresis on SDS-PAGE (7%), thentransferred electrophoretically overnight to nitrocellulose paper(Towbin, H., et al., [1979] Proc. Nat. Acad. Sci. 76(9): 4350-4379). Thenitrocellulose paper is soaked for one hour at room temperature in 0.5%non-fat dry milk dissolved in phosphate buffered saline, and furthersoaked overnight at 4° C. in blocking solution (2% bovine serum albuminin 10 mM Tris/0.15 M NaCl/0.1% sodium azide, pH 7.4). The nitrocellulosemembranes are incubated with antibodies (IgG factions of anti-TGF-beta,anti-TGF-alpha, anti-EGF and anti-bFGF) purified by protein A (1×10⁶cpm/ml) in blocking buffer for 1 hour. The membrane is washed with PBScontaining Nonidet P-40 between incubations. X-O-mat AR2 film (Kodak) isexposed to the membrane at −70° C. and films are developed to examinethe expression of growth factors.

[0041] Determination of c-fos and c-myc Expression

[0042] Expression of c-fos and c-myc in treated and untreated prostatictissue is determined by Northern blot analysis as follows. Tissue ishomogenized in lysis buffer for 15 seconds or until the tissuehomogenizes. Sodium acetate is added and the solution is mixed byswirling. An equal volume of water-saturated phenol is added and mixedby inversion, followed by addition of chloroform/isoamyl alcohol. Thesolution is vortexed vigorously for 30 seconds, and allowed to settle onice for 15 minutes. The solution is centrifuged for 10-20 minutes at 4°C. After centrifugation, the aqueous phase is carefully aspirated andplaced in a new polypropylene tube. One volume of isopropanol is addedand the solution is mixed by swirling. The solution is placed in a −20°C. freezer for at least 60 minutes to precipitate RNA. Afterprecipitation, the tube is centrifuged for 10 minutes, and thesupernatant is decanted, leaving the RNA pellet. One ml of ethanol isadded, and the tube is centrifuged for an additional 10 minutes. Theaqueous phase is discarded, and the pellet is washed with 100% ethanolby vortexing. The RNA pellet is redissolved in 0.4 ml of lysis buffer.The RNA is reprecipitated by the addition of 100% ethanol and incubationat −20° C. freezer for at least 60 minutes. The solution is centrifugedand the supernatant discarded. RNA concentration is determined bydiluting 5 μL of sample into 995 μL of DEPC water and measuring theratio of absorbance at 260/280 nm.

[0043] The following examples are provided by way of describing specificembodiments without intending to limit the scope of the invention in anyway.

EXAMPLE 1

[0044] Denervation of the Prostate

[0045] Unilateral denervation of the prostate is carried out by removalof the pelvic ganglia, which overlie the prostate of the rat. Thisapproach preserves the functional integrity of the bladder and posteriorurethra and removes the possibility for artifact arising from majordisturbances in blood flow or micturation. Control animals undergo shamoperations without concurrent denervation of the prostate. Afterdenervation, the animals are allowed to recover and maintained prior tocollection of the prostate. The prostate is preserved, prepared forlight microscopy and examined histologically. The major findings are (1)reduced epithelial cell height primarily due to a decrease in the clearsupranuclear zone (due to a reduction in the amount and size of theapical cisternae and the endoplasmic reticulum); (2) major changes inprotein expression on SDS gel electrophoresis (the endoplasmic reticulumis important in protein synthesis) (3) modest reduction in the number ofsecretory granules; (4) an increase in intracellular vacuoles,intercellular empty spaces and reduction in microvilli on the cellsurface; and (5) a significant increase in nerve growth factor (NGF)content ipsilateral to the denervation relative to the control group(188±10 vs. 46±20 vs. 29±16 pg/g wet tissue (±SD) NGF is known toinfluence only sympathetic and sensory neurons. N=15 in both the controland experimental groups.

EXAMPLE 2

[0046] Effect of Neurotoxin Injection on Normal Prostate: Rat Prostate

[0047] Rats were randomly assigned into three groups. The first groupreceived a single acute dose of Botulinum toxin type A (Botox, Allergen)of 5, 10 or 15 IU. These animals were sacrificed one week afterinjection. The second group received a series of 4 weekly injections of5 IU of Botulinum toxin and were sacrificed at 5 weeks. Control ratsreceived saline injections. Injections were performed as single orserial injections into the left and/or right ventral lobe of theprostate. Note that an injection of methylene blue into one lobe of therat prostate showed immediate diffusion into the opposite lobe. Thus,there was communication between the prostate lobes and therefore thecontralateral lobe could not be used as a true comparative control.

[0048] The weight of each prostate ventral lobe collected from healthyanimals was approximately 0.50 gram. All toxin-treated animals showedshrinkage of prostate volume, first in the injected lobe, and withsubsequent injections, reduction in the overall volume. After fourserial injections, the left prostate lobe weighed 0.12-0.17 gram, whilethe right lobe weighed 0.10-0.14 grams. This represented a reduction ofover two-thirds of the original size.

EXAMPLE 3

[0049] Effect of Neurotoxin Injection on Urological Dysfunctions: HumanData

[0050] Three patients with recalcitrant voiding dysfunction were treatedwith injections of botulinum toxin (Botox) as follows. Patient 1 was a47-year-old male who was incontinent secondary to an injury sustained atthe cervical vertebrae (level C6-C7) sustained 14 months previously.Urodynamics on presentation revealed a bladder capacity of 30 cc and aweak sphincter (peak urethral pressure of 40 cm water). He had failedmultiple pharmacological regimes and was intolerant to penileclamp/condom devices.

[0051] He received four weekly 200 IU botulinum toxin injections intothe bladder neck for total dose of 800 IU. Post-injection, his bladdercapacities ranged from 300-400 cc with oxybutinin and 150-200 cc withoutoxybutinin. Peak bladder pressures pre-injection had been 200-cm water,compared to post injection bladder pressures of 40 cm of water. Thepatient was continent with a penile clamp after treatment with botulinumtoxin. In addition, walking and erections improved due to reducedbladder spasticity.

[0052] Patient 2 was at 55 year old T12 paraparetic female secondary totraumatic injury 14 years previous. The patient presented with urgeincontinence, and had been on self-catheterization every 2 hours duringthe day and two times at night. The patient received injections into thelateral bladder wall in two weekly injections of 200 IU each for a totalof 400 IU of botulinum toxin. The patient's voiding diary data revealedpre-injection capacities of between 150-200 cc. Post injection, diarydata indicated bladder capacity increased to 300-400 cc. In addition,the patient no longer had annoying constant urge type dysfunction, sleptthrough the night and was continent on self-catheterization every 4hours.

[0053] Patient 3 was a 65 year old male with disabling perineal painfollowing radiation treatment for prostatic cancer. The patient hadfailed medical therapy. He was treated with one 200 IU injection ofbotulinum toxin into the external urethral sphincter. The patientexperienced a dramatic relief of testicle pain and had far less severepain in the shaft of the penis. Erections were not affected.

EXAMPLE 4

[0054] Determination of Smallest Effective Dose

[0055] Rats are injected in the prostate ventral lobes with single andserial doses of botulinum toxin (Botox) The prostates are harvested atdifferent time intervals to determine the smallest effective dose, aswell as the morphological and physiological changes taking place withtime. The smallest effective dose is defined as that dose that woulddemonstrate a decrease in prostate volume.

[0056] To assess the response to electrical field stimulation,preparations are mounted between two platinum electrodes placed in theorgan bath. The tension of the preparations is adjusted. Transmuralstimulation of nerves is performed using a Danted Neuromatic 2000Stimulator delivering single square-wave pulses at supramaximal voltagewith a duration of 0.8 milliseconds at a frequency of 0.5 to 80 hertz.The polarity of the electrodes is changed after each pulse by means of apolarity-changing unit. The train duration is five seconds and the traininterval 120 seconds. Isometric tension is recorded by using a Gouldthermo-array 8-channel recorder. Separate experiments are performed todetermine the preload tension producing optimal responses. In addition,the effect of the electric field stimulation in the presence ofdifferent concentrations of individual neuropeptides is determined.These neuropeptides are 10-20 μM adrenaline, 10 μM clonidine, 5-50 mMregitine, 10 nM-0.1 μM acetylcholine, 1-3 μM atropine, 1 nM-10 μMnifedipine, 1-10 nM VIP and 1-250 nM NPY. The effect of nitroprusside (anitric oxide releasing substance) and methylene blue (a guanylatecyclase inhibitor) on prostate tone and contraction resulting from fieldstimulation also is examined in these tissues.

EXAMPLE 5

[0057] Effect of Botulinum Toxin on Rat Prostatic Tissue: Comparison ofHormonally Intact Rats to Hormonally Deprived Rats

[0058] To determine if there is any interaction between the neurotoxinand testicularly-derived hormones, studies are performed which willexamine the interaction of the neurotoxin with hormonal components.These studies will compare prostatic tissue treated with botulinum toxinharvested from rats that have undergone orchiectomy (hormonally depletedrats) and prostatic tissue from rats treated with botulinum toxin thatdid not undergo orchiectomy. Fifty-two age-matched rats are treated asdescribed below. Four healthy rats will undergo a sham operationconsisting of anesthesia induction, exposure of the prostate andinjection of 0.2 cc saline into the left ventral lobe of the prostate.Three rats are given bilateral orchiectomy with no injection to theprostate (hormonally depleted controls), five rats will have orchiectomyand injection of 0.2 ml saline in the left ventral lobe (hormonaldepletion+surgical stress control). Four groups of rats receivebotulinum injections of 0.5 IU, 1.0 IU, 1.5 IU and 2.5 IU only(hormonally intact experimental rats). Sixteen rats undergo bilateralorchiectomy. Eight of these rats are treated with a single injection of2.5 IU botulinum toxin into the left ventral lobe 5 weeks after surgery.All rats are sacrificed after six weeks, and the harvested prostate isprepared for examination as described above. A similar atrophic effecton glandular epithelium is expected.

EXAMPLE 6

[0059] Effects of Botulinum Toxin on Patients

[0060] Patients affected by benign prostatic hyperplasia, abacterialprostatis, or prostatodynia are studied both before and after treatmentwith botulinum toxin. Patients are eligible for inclusion in this studyif they are affected by BPH between the ages of 40 and 80, or if theyare between 25 and 60 and have been diagnosed with abacterialprostatitis or prostatodynia. Preferred patients are those who are notgood surgical candidates. Patients are evaluated prior to treatment bydetermination of prostate specific antigen levels (PSA), evaluation ofurodynamic parameters (cystometrogram, urethral pressure profile andflowmetry), determination of American Urological Association (AUA)symptom score (Barry, M. J., et al., [1992] J. Urol., 148: 1549-1557),maintenance of a voiding diary, and examination of the prostate bytransrectal ultrasound with biopsy (for BPH patients only). One weekafter initial evaluation is completed, the patient is injectedurethroscopically with 200 IU of botulinum toxin as either singleunilateral injections, serial unilateral injections or bilateralinjections. BPH patients are treated by TURP or undergo controlTURP-biopsy 7 days after single injection or 5 weeks after serialinjections. The harvested prostatic tissues are prepared for examinationas described in Examples 1, 2, 3, and 7-10. The patients arere-evaluated after injection using the same parameters examined duringthe initial evaluation.

[0061] The foregoing description of the invention is exemplary forpurposes of illustration and explanation. It will be apparent to thoseskilled in the art that changes and modifications are possible withoutdeparting from the spirit and scope of the invention. All documentscited herein are hereby incorporated by reference. It is intended thatthe following claims be interpreted to embrace all such changes andmodifications.

What is claimed is:
 1. A method of treating neurological-urologicalconditions in mammals comprising the step of administering atherapeutically effective amount of at least one neurotoxin. I. Themethod according to claim 1, wherein the neurological-urologicalcondition includes prostatic enlargement. II. The method according toclaim 2, wherein the mammal is human male.
 4. The method of claim 2,wherein the prostatic enlargement is selected from the group consistingof benign prostatic hyperplasia, abacterial prostatitis andprostatodynia.
 5. The method of claim 1, wherein the neurotoxin isselected from the group consisting of botulinum toxin, capsaicin,terodotoxin, resinoferatoxin and α-bungotoxin. The method of claim 5,wherein the neurotoxin is Botulinum toxin type A. III. The method ofclaim 5, wherein the Botulinum toxin type A is Botox. IV. The method ofclaim 2, wherein the prostatic enlargement is benign prostatichyperplasia. V. The method of claim 1, wherein theneurological-urological condition is selected from the group consistingof pelvic pain (e.g., interstitial cystitis, endometriosis,prostatodynia, urethral instability syndromes), pelvic myofascialelements (e.g., levator sphincter, dysmenorrhea, anal fistula,hemorrhoid), urinary incontinence (e.g., unstable bladder, unstablesphincter), prostate disorders (e.g., BPH, prostate cancer), recurrentinfection (secondary to sphincter spasticity), and urinary retention(secondary to spastic sphincter, hypertrophied bladder neck, andneurogenic bladder dysfunction (e.g., Parkinson's Disease, spinal cordinjury, stroke, multiple sclerosis, spasm reflex) and other suchurological conditions of a nervous etiology.